Figure 3 from SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation–Phosphorylation Cross-talk

Abstract

<p>SMYD3–RNF113A methylation signaling in SCLC cell lines. <b>A,</b> Immunodetection of endogenous RNF113A K20me3 following immunoprecipitation of total RNF113A in SCLC H69 cells transduced with shRNA targeting <i>SMYD3</i> (sh<i>SMYD3</i>) and a control nontargeting shRNA (shControl). Tubulin was used as a loading control. <b>B,</b> Immunoblot analysis with indicated antibodies as in <b>A</b> of H1048 SCLC cells expressing doxycycline-inducible sh<i>SMYD3</i> or treated with SMYD3i (EPZ031686). Tubulin was used as a loading control. <b>C,</b> Immunoblot analysis with indicated antibodies using lysates obtained from human SCLC cell lines representing all four molecular subtypes (NAPY) classified by expression of specific markers (NEUROD1<sup>+</sup>; ASCL1<sup>+</sup>; POU2F3<sup>+</sup>; YAP1<sup>+</sup>). GAPDH was used as a loading control. <b>D,</b><i>SMYD3</i> and <i>RNF113A</i> expression in human samples representing different molecular SCLC subtypes. Boxes represent 25th to 75th percentiles; whiskers: 10% to 90%; center line: median. <i>P</i> values were calculated by the Kruskal–Wallis test. Analyses were performed using FPKM data for each specified gene obtained from ref. <a href="#bib34" target="_blank">34</a>. NAPY SCLC subclassification was based on the original classification from ref. <a href="#bib32" target="_blank">32</a>. <b>E,</b> Analysis of DMS-114 SCLC cell line growth response to increasing concentrations of 4H-CP. Cells were transduced with doxycycline-inducible sh<i>SMYD3</i> and complemented with the expression of RNF113A or both SMYD3 and RNF113A. The percentage of viable cells under each condition was normalized to vehicle-treated (control) cells. Each condition represents the mean of three technical replicates from two independent experiments. <i>P</i> values were calculated by two-way ANOVA with the Tukey test for multiple comparisons. Data are represented as nonlinear regression with mean ± SEM. In all panels, representative of at least three independent experiments is shown unless stated otherwise. The numbers below the immunoblot lines represent the relative signal quantification (see also Supplementary Table S5).</p>

Related Publications